Complex interplays between phytosterols and plastid development

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs. Despite their variety of structures, all isoprenoids derive from the common C₅ precursor isopentenyl diphosphate (IPP). Plants synthesize IPP through two different metabolic pathways, the mevalonic acid (MVA) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways that operate in the cytosol-RE and plastids, respectively. MEP-derived isoprenoids include important compounds for chloroplast function and as such, knock-out mutant plants affected in different steps of this pathway display important alterations in plastid structure. These alterations often lead to albino phenotypes and lethality at seedling stage. MVA knock-out mutant plants show, on the contrary, lethal phenotypes already exhibited at the gametophyte or embryo developmental stage. However, the recent characterization of conditional knock-down mutant plants of farnesyl diphosphate synthase (FPS), a central enzyme in cytosolic and mitochondrial isoprenoid biosynthesis, revealed an unexpected role of this pathway in chloroplast development and plastidial isoprenoid metabolism in post-embryonic stages. Upon FPS silencing, chloroplast structure is severely altered, together with a strong reduction in the levels of MEP pathway-derived major end products. This phenotype is associated to misregulation of genes involved in stress responses predominantly belonging to JA and Fe homeostasis pathways. Transcriptomic experiments and analysis of recent literature indicate that sterols are the cause of the observed alterations through an as yet undiscovered mechanism

Nerolidol production in agroinfiltrated tobacco: Impact of protein stability and membrane targeting of strawberry (Fragraria ananassa) NEROLIDOL SYNTHASE1

The sesquiterpene alcohol nerolidol, synthesized from farnesyl diphosphate (FDP), mediates plant-insect inter- actions across multiple trophic levels with major implications for pest management in agriculture. We compared nerolidol engineering strategies in tobacco using agroinfiltration to transiently express strawberry (Fragraria ananassa) linalool/nerolidol synthase (FaNES1) either at the endoplasmic reticulum (ER) or in the cytosol as a soluble protein. Using solid phase microextraction and gas chromatography-mass spectrometry (SPME-GCMS), we have determined that FaNES1 directed to the ER via fusion to the transmembrane domain of squalene synthase or hydroxymethylglutaryl - CoA reductase displayed significant improvements in terms of transcript levels, protein accumulation, and volatile production when compared to its cytosolic form. However, the highest levels of nerolidol production were observed when FaNES1 was fused to GFP and expressed in the cytosol. This SPME-GCMS method afforded a limit of detection and quantification of 1.54 and 5.13 pg, respectively. Nerolidol production levels, which ranged from 0.5 to 3.0 μg/g F.W., correlated more strongly to the accumulation of recombinant protein than transcript level, the former being highest in FaNES-GFP transfected plants. These results indicate that while the ER may represent an enriched source of FDP that can be exploited in metabolic engineering, protein accumulation is a better predictor of sesquiterpene production

Identificación de interactores del regulador de la homeostasis lipídica AtArv1: caracterización del factor de transcripción anclado a membrana maMYB de Arabidopsis

[spa] Los estudios realizados sobre la proteína Arv1 en levaduras y animales indican que estaría involucrada en el mantenimiento de la homeostasis lipídica intracelular, aunque se desconoce su función bioquímica concreta. A. thaliana posee los genes ARV AtARV1 y AtARV2, que codifican las proteínas funcionales AtArv1 y AtArv2, respectivamente, ancladas a la membrana del RE. Partiendo de la hipótesis de que las proteínas AtArv interaccionan con otras proteínas para poder ejercer su función biológica, este trabajo se inició con el cribado de un banco de cDNA de plántulas de Arabidopsis empleando AtArv1 como cebo y la técnica de doble híbrido en levadura (MbYTH), donde se identificó que la proteína maMYB interacciona con AtArv1. La proteína maMYB, codificada por un único gen en Arabidopsis, tiene características de un MTTF de la familia MYB, puesto que contiene un dominio citosólico R2R3-MYB en la región C-terminal y dos DTMs en la región N-terminal. En este sentido, se determinó que maMYB se localiza en las membranas del RE y presenta ambos extremos proyectados hacia el citosol. Además, se demostró que la versión truncada maMYB91-309, que contiene el dominio citosólico R2R3-MYB, se localiza en el núcleo y preferentemente en el nucléolo. El análisis del patrón de expresión de la proteína maMYB reveló que lo hace mayoritariamente en raíz y parte aérea de plántulas de Arabidopsis. Empleando una estrategia de silenciamiento inducible basada en el uso de microRNAs artificiales (amiRNA), se obtuvieron líneas mutantes de maMYB condicionales en donde se observó una reducción pronunciada en los niveles de mRNA y proteína maMYB y una alteración severa del desarrollo de las plántulas de Arabidopsis, que se caracterizó por una reducción del tamaño general de la plántula, una disminución de la longitud de la raíz primaria, una inhibición de la formación de raíces laterales y pelos radiculares, una alteración en la forma, el tamaño y el número de las células de las diferentes capas de la raíz, una desestructuración de las células epiteliales de los cotiledones y una alteración en los niveles de los productos finales de la ruta de esteroles y de sus precursores inmediatos. Mediante el estudio de la expresión génica global con la tecnología RNA‐seq se determinó que el silenciamiento de maMYB provoca la desregulación de grupos de genes involucrados en el desarrollo y el crecimiento de la planta, que son coherentes con el fenotipo observado. Tanto en la raíz como en la parte aérea se identificaron genes desregulados involucrados en la elongación celular y relacionados con la pared celular. En la raíz se identificaron genes desregulados relacionados con la síntesis de chalconas, el transporte de auxinas y la morfogénesis de los pelos radiculares. En la parte aérea se identificaron genes desregulados implicados en la síntesis de fenilpropanoides y auxinas y la respuesta a auxinas. Además, los genes desregulados como consecuencia del silenciamiento de maMYB presentan un alto enriquecimiento de genes regulados epigenéticamente a través de la trimetilación de la lisina 27 de la histona H3 (H3K27me3), lo que sugiere que maMYB podría estar relacionado con este proceso necesario para el correcto desarrollo de la planta. Por otro lado, se observó que la sobreexpresión de maMYB91-309 revierte (en mayor o menor medida) el fenotipo causado por el silenciamiento de maMYB, lo cual avala la funcionalidad in vivo del dominio citosólico y el papel de maMYB como MTTF, cuyo dominio R2R3-MYB citosólico necesitaría liberarse por acción de proteasas intramembrana y trasladarse al núcleo donde ejercería su función. Las alteraciones observadas en el desarrollo de los mutantes de silenciamiento de maMYB sugieren que éste sea un MTTF que se libere en respuesta a estímulos de desarrollo.[eng] Cerca avançada Restringir a TDX Inici | Què és? | Preguntes més freqüents (FAQ) | Contacte English | Castellano Consultar TDX Per universitats i departaments Per data de defensa Per autors/directors Per títols Per matèries Consultar departament Per data de defensa Per autors/directors Per títols Per matèries Estadístiques Per tesi Per departament Per universitat Tot TDX El meu TDX Registrat com [email protected] (Finalitza la sessió) Perfil Enviaments Alertes per correu-e Opcions administrador Edita aquest element Altres portals de tesis Tesis europees Tesis internacionals Novetats Pàgina inicial del TDX > Universitat de Barcelona > Departament de Bioquímica i Biologia Molecular (Farmàcia) > Visualitza tesi Logotip de la col·lecció Empreu aquest identificador per citar o enllaçar aquesta tesi: http://hdl.handle.net/10803/285488 Títol: Identificación de interactores del regulador de la homeostasis lipídica AtArv1: caracterización del factor de transcripción anclado a membrana maMYB de Arabidopsis Autor/a: Caudepón Giménez, Daniel Director/a: Ferrer i Prats, Albert Arró i Plans, Montserrat Departament/Institut: Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Farmàcia) Resumen: Los estudios realizados sobre la proteína Arv1 en levaduras y animales indican que estaría involucrada en el mantenimiento de la homeostasis lipídica intracelular, aunque se desconoce su función bioquímica concreta. A. thaliana posee los genes ARV AtARV1 y AtARV2, que codifican las proteínas funcionales AtArv1 y AtArv2, respectivamente, ancladas a la membrana del RE. Partiendo de la hipótesis de que las proteínas AtArv interaccionan con otras proteínas para poder ejercer su función biológica, … [+] Abstract: In yeast and animals, Arv1 protein is involved in the intracellular lipid homeostasis, although its particular biochemistry function remains unknown. A. thaliana has the ARV genes AtARV1 and AtARV2, which codify the functional proteins AtArv1 and AtArv2, respectively, tethered to the ER membrane. Assuming that AtArv proteins need to interact with other proteins to carry out its biological function, this work was initiated with a screening of a cDNA library from Arabidopsis seedlings using AtArv1 as a bait and the yeast two hybrid version MbYTH, where we identified maMYB as an interactor of AtArv1. maMYB protein, codified by a unique gene in Arabidopsis, has characteristics of MYB family MTTF, since it contains a citosolic R2R3-MYB domain at the C-terminal region and two TMDs at the N-terminal region. In this way, we demonstrated that maMYB is localized in the ER membrane facing both extremes to the citosol, and the truncated form maMYB91-309, which contains the R2R3-MYB domain, is localized in the nucleus and preferentially in the nucleolus. The analysis of the expression pattern of maMYB protein revealed that the highest levels were found in the root and the shoot of Arabidopsis seedlings. Using an inducible silencing strategy based on the artificial microRNAs (amiRNAs) technology, we obtained maMYB inducible silencing mutants that showed a strong alteration in the root and shoot development of Arabidopsis seedlings. Through a global expression study using RNA-seq technology, we determined that maMYB silencing causes the deregulation of gene groups involved in plant development and growth, which is coherent with the observed phenotype. Interestingly, we observed that the maMYB91-309 overexpression revert (at more or less extent) the phenotype caused by maMYB silencing, which support the functionality of the citosolic R2R3-MYB domain in vivo and the role of maMYB as a MTTF, whose R2R3-MYB domain may be released by the action of intramembrane proteases and be transported to the nucleus where it may develop its function. The developmental alterations observed in the maMYB silencing mutants suggest that maMYB might be a MTTF released in respond to developmental cues

Identificación de interactores del regulador de la homeostasis lipídica AtArv1: caracterización del factor de transcripción anclado a membrana maMYB de Arabidopsis

Los estudios realizados sobre la proteína Arv1 en levaduras y animales indican que estaría involucrada en el mantenimiento de la homeostasis lipídica intracelular, aunque se desconoce su función bioquímica concreta. A. thaliana posee los genes ARV AtARV1 y AtARV2, que codifican las proteínas funcionales AtArv1 y AtArv2, respectivamente, ancladas a la membrana del RE. Partiendo de la hipótesis de que las proteínas AtArv interaccionan con otras proteínas para poder ejercer su función biológica, este trabajo se inició con el cribado de un banco de cDNA de plántulas de Arabidopsis empleando AtArv1 como cebo y la técnica de doble híbrido en levadura (MbYTH), donde se identificó que la proteína maMYB interacciona con AtArv1. La proteína maMYB, codificada por un único gen en Arabidopsis, tiene características de un MTTF de la familia MYB, puesto que contiene un dominio citosólico R2R3-MYB en la región C-terminal y dos DTMs en la región N-terminal. En este sentido, se determinó que maMYB se localiza en las membranas del RE y presenta ambos extremos proyectados hacia el citosol. Además, se demostró que la versión truncada maMYB91-309, que contiene el dominio citosólico R2R3-MYB, se localiza en el núcleo y preferentemente en el nucléolo. El análisis del patrón de expresión de la proteína maMYB reveló que lo hace mayoritariamente en raíz y parte aérea de plántulas de Arabidopsis. Empleando una estrategia de silenciamiento inducible basada en el uso de microRNAs artificiales (amiRNA), se obtuvieron líneas mutantes de maMYB condicionales en donde se observó una reducción pronunciada en los niveles de mRNA y proteína maMYB y una alteración severa del desarrollo de las plántulas de Arabidopsis, que se caracterizó por una reducción del tamaño general de la plántula, una disminución de la longitud de la raíz primaria, una inhibición de la formación de raíces laterales y pelos radiculares, una alteración en la forma, el tamaño y el número de las células de las diferentes capas de la raíz, una desestructuración de las células epiteliales de los cotiledones y una alteración en los niveles de los productos finales de la ruta de esteroles y de sus precursores inmediatos. Mediante el estudio de la expresión génica global con la tecnología RNA‐seq se determinó que el silenciamiento de maMYB provoca la desregulación de grupos de genes involucrados en el desarrollo y el crecimiento de la planta, que son coherentes con el fenotipo observado. Tanto en la raíz como en la parte aérea se identificaron genes desregulados involucrados en la elongación celular y relacionados con la pared celular. En la raíz se identificaron genes desregulados relacionados con la síntesis de chalconas, el transporte de auxinas y la morfogénesis de los pelos radiculares. En la parte aérea se identificaron genes desregulados implicados en la síntesis de fenilpropanoides y auxinas y la respuesta a auxinas. Además, los genes desregulados como consecuencia del silenciamiento de maMYB presentan un alto enriquecimiento de genes regulados epigenéticamente a través de la trimetilación de la lisina 27 de la histona H3 (H3K27me3), lo que sugiere que maMYB podría estar relacionado con este proceso necesario para el correcto desarrollo de la planta. Por otro lado, se observó que la sobreexpresión de maMYB91-309 revierte (en mayor o menor medida) el fenotipo causado por el silenciamiento de maMYB, lo cual avala la funcionalidad in vivo del dominio citosólico y el papel de maMYB como MTTF, cuyo dominio R2R3-MYB citosólico necesitaría liberarse por acción de proteasas intramembrana y trasladarse al núcleo donde ejercería su función. Las alteraciones observadas en el desarrollo de los mutantes de silenciamiento de maMYB sugieren que éste sea un MTTF que se libere en respuesta a estímulos de desarrollo.In yeast and animals, Arv1 protein is involved in the intracellular lipid homeostasis, although its particular biochemistry function remains unknown. A. thaliana has the ARV genes AtARV1 and AtARV2, which codify the functional proteins AtArv1 and AtArv2, respectively, tethered to the ER membrane. Assuming that AtArv proteins need to interact with other proteins to carry out its biological function, this work was initiated with a screening of a cDNA library from Arabidopsis seedlings using AtArv1 as a bait and the yeast two hybrid version MbYTH, where we identified maMYB as an interactor of AtArv1. maMYB protein, codified by a unique gene in Arabidopsis, has characteristics of MYB family MTTF, since it contains a citosolic R2R3-MYB domain at the C-terminal region and two TMDs at the N-terminal region. In this way, we demonstrated that maMYB is localized in the ER membrane facing both extremes to the citosol, and the truncated form maMYB91-309, which contains the R2R3-MYB domain, is localized in the nucleus and preferentially in the nucleolus. The analysis of the expression pattern of maMYB protein revealed that the highest levels were found in the root and the shoot of Arabidopsis seedlings. Using an inducible silencing strategy based on the artificial microRNAs (amiRNAs) technology, we obtained maMYB inducible silencing mutants that showed a strong alteration in the root and shoot development of Arabidopsis seedlings. Through a global expression study using RNA-seq technology, we determined that maMYB silencing causes the deregulation of gene groups involved in plant development and growth, which is coherent with the observed phenotype. Interestingly, we observed that the maMYB91-309 overexpression revert (at more or less extent) the phenotype caused by maMYB silencing, which support the functionality of the citosolic R2R3-MYB domain in vivo and the role of maMYB as a MTTF, whose R2R3-MYB domain may be released by the action of intramembrane proteases and be transported to the nucleus where it may develop its function. The developmental alterations observed in the maMYB silencing mutants suggest that maMYB might be a MTTF released in respond to developmental cues

Suppressing farnesyl diphosphate synthase alters chloroplast development and triggers sterol-dependent induction of jasmonate- and Fe-related responses

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development.We acknowledge financial support from the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533). This work was supported by the Spanish Government (grant nos. BIO2009–06984 and AGL2013–43522-R to A.F.) and the Generalitat de Catalunya (grant no. 2014SGR–1434).Peer reviewe

Complex interplays between phytosterols and plastid development

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs. Despite their variety of structures, all isoprenoids derive from the common C₅ precursor isopentenyl diphosphate (IPP). Plants synthesize IPP through two different metabolic pathways, the mevalonic acid (MVA) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways that operate in the cytosol-RE and plastids, respectively. MEP-derived isoprenoids include important compounds for chloroplast function and as such, knock-out mutant plants affected in different steps of this pathway display important alterations in plastid structure. These alterations often lead to albino phenotypes and lethality at seedling stage. MVA knock-out mutant plants show, on the contrary, lethal phenotypes already exhibited at the gametophyte or embryo developmental stage. However, the recent characterization of conditional knock-down mutant plants of farnesyl diphosphate synthase (FPS), a central enzyme in cytosolic and mitochondrial isoprenoid biosynthesis, revealed an unexpected role of this pathway in chloroplast development and plastidial isoprenoid metabolism in post-embryonic stages. Upon FPS silencing, chloroplast structure is severely altered, together with a strong reduction in the levels of MEP pathway-derived major end products. This phenotype is associated to misregulation of genes involved in stress responses predominantly belonging to JA and Fe homeostasis pathways. Transcriptomic experiments and analysis of recent literature indicate that sterols are the cause of the observed alterations through an as yet undiscovered mechanism

Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed

Thermal stability of FPS1S and FPS2 enzyme activity.

<p>(A) Activity of purified FPS1S (open symbols) and FPS2 (closed symbols) was measured after incubation at either 37°C (squares) or 45°C (circles) for the indicated time periods. (B) FPS activity in 16,000 <i>g</i> protein extracts from <i>fps1-1</i> (FPS2 activity, closed circles) and <i>fps2-1</i> (FPS1 activity, open circles) mutants was determined after incubation at 45°C for the indicated times. In both cases enzyme activities are expressed relative to the FPS activity values at time 0 min and the mean values and SE were calculated from three independent experiments. (C) Differential scanning fluorimetry (DSF) results plotted as change in fluorescence emission intensity (normalized to unity at its maximum) with increasing temperature (20–80°C). The FPS1S and FPS2 curves correspond to 6 µM enzyme.</p

Characterization of <i>fps2-1</i> mutant lines harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> genes.

<p>(A) The expression of <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> was investigated using total RNA from 12-day-old seedlings of Arabidopsis wild-type, <i>fps2-1</i> and the indicated lines of the <i>fps2-1</i> mutant harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> chimeric genes. PCR products were electrophoresed in a 1% agarose gel. The size in bp of the amplified cDNA fragments corresponding to <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> (1088 bp) and <i>PP2A</i> genes (307 bp) is indicated on the right. Numbers on the left indicate the sizes in bp of DNA markers shown in lane M. (B) Western blot analysis of total FPS protein in 16,000 <i>g</i> extracts from seeds of plant lines indicated above (upper panel). The lower panel shows the Coomassie blue-stained electrophoretic protein patterns in the 35 to 50 kDa range of extracts used for FPS protein level determinations. Images show the results of one representative experiment. (C) FPS activity in the 16,000 <i>g</i> protein extracts used for Western blot analysis. FPS activity in mutants is expressed relative to that in the wild-type, which was assigned a value of 100. The mean values and SE were calculated from three independent experiments.</p

Expression in <i>E. coli</i> and purification of recombinant FPS1S and FPS2 proteins.

<p>(A) Total protein extracts from <i>E. coli</i> cells harbouring either pGEX-3X-NotI-FPS1 or pGEX-3X-NotI-FPS2 before (lanes 1 and 3) and after induction (lanes 2 and 4) of GST-FPS1S and GST-FPS2 expression with 0.4 mM IPTG for 6 hours at 22°C. (B) Soluble protein extracts of IPTG-induced <i>E. coli</i> cells harbouring either pGEX-3X-NotI-FPS1 (lane 1) or pGEX-3X-NotI-FPS2 (lane 2), and purified native FPS1S (lane 3) and FPS2 (lane 4) protein preparations after Glutathione-Sepharose 4B affinity column chromatography, proteolytic digestion with Factor Xa and protease removal. Arrows indicate the position of GST-FPS protein fusions and purified native FPS proteins. Molecular masses of standards (M) are indicated in kDa.</p